Purification and characterization of Aspergillus β-d-galactanases acting on β-1,4- and β-1,3/6-linked arabinogalactans

Luonteri, Elina; Laine, Christiane; Uusitalo, Sanna; Teleman, Anita; Siika‐aho, Matti; Tenkanen, Maija

doi:10.1016/s0144-8617(02)00303-x

Cited by 32 publications

(24 citation statements)

References 43 publications

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“…The lack of the necessary enzyme for hydrolyzing 1,3-linked galactan (Luonteri et al 2003) would also support the theory for long chains of 1,3/6-linked galactan in RLCCs. A second possibility is that shorter chains of galactose units are connected to lignin at the reducing end.…”

Section: Possible Lignin-carbohydrate Binding Sitessupporting

confidence: 61%

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Carbohydrate structures in residual lignin-carbohydrate complexes of spruce and pine pulp

2004

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Residual lignin carbohydrate complexes (RLCC) were isolated enzymatically from spruce and pine pulp. The RLCCs contained 4.9-9.4% carbohydrates, with an enrichment of galactose and arabinose compared to the original pulp samples. The main carbohydrate units present in all studied RLCCs were 4-substituted xylose, 4-, 3-and 3,6-substituted galactose, 4-substituted glucose and 4 and 4,6-substituted mannose. These units were assigned to carbohydrate residues of xylan, 1,4-and 1,3/ 6-linked galactan, cellulose and glucomannan.RLCCs of surface material and the inner part of spruce kraft pulp fiber were compared to obtain information on the heterogeneity of layers of the fiber wall. The 1,4-linked galactan was the major galactan in RLCC of fiber surface material of spruce kraft pulp. Towards the inner part of the fiber, the proportion of 1,3/6-linked galactan increased relative to 1,4-linked galactan. This finding is presented for the first time. 1,3/6-Linked galactan structures are suggested to have a role in restricting lignin removal from the secondary fiber wall.RLCCs of three different alkaline pine pulps were studied before and after oxygen delignification to evaluate differences resulting from the cooking method. The pulps were conventional kraft pine pulp (PCK), a polysulfide/ anthraquinone pine pulp (PPSAQ) and a soda/anthraquinone pine pulp (PSoAQ); all were cooked to approximately kappa number 30. Small differences were found in the carbohydrate structures of the unbleached pulps. The study indicated that the RLCC of unbleached PSoAQ pulp contained longer oligomeric carbohydrate chains and less branched 1,3/6-linked galactan residues than the RLCCs of unbleached PCK and PPSAQ pulps. The RLCC of the unbleached PSoAQ also contained more 1,4-linked glucose units suggesting a greater number of linkages of lignin to cellulose in the PSoAQ pulp than in the other two pulps. All RLCCs of oxygen-delignified pulps had more non-reducing ends and less 1,3/ 6-linked galactan than the corresponding RLCCs of the unbleached pulps. The RLCC of the oxygen-delignified PSoAQ pulp had a higher ratio of 1,4-galactan to 1,3/6-linked galactan and shorter xylan residues than the RLCCs of oxygen-delignified PCK and PPSAQ pulps.

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Section: Possible Lignin-carbohydrate Binding Sitessupporting

confidence: 61%

“…These positive results could probably be further improved by using an endo-b-1,3-galactanase simultaneously (Tenkanen et al 2002). However, such an enzyme has not been found (Luonteri et al 2003). Arabinose was present mainly as a 5-substituted unit ( Figure 3A).…”

Section: Rlccs Of Spruce Kraft Pulp -Surface Materials Compared To Peementioning

confidence: 98%

Carbohydrate structures in residual lignin-carbohydrate complexes of spruce and pine pulp

2004

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“…A comparable reaction may also be occurring in some fungi. In comparison to bacterial galactanases, most fungal galactanases (5,11,39,45,52,68) yield ␤-1-4-galactobiose and some fungi have ␤-galactosidases with reasonable K m values (4.5 to 19.4 mM) on galactobiose (46). The association between GHF 53 and GHF 2 genes in a few bacteria also supports possible GHF 53-GHF 2 synergy.…”

Section: Vol 72 2006 Functions For Ghf 42 ␤-Galactosidases 7735mentioning

confidence: 85%

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Shipkowski

Brenchley

2006

Appl Environ Microbiol

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Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the ␤-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased ␤-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 ␤-galactosidases. Similar functions for other ␤-galactosidases in both GHFs 2 and 42 are suggested by genomic data.␤-Galactosidases, such as the LacZ ␤-galactosidase of Escherichia coli, are used as molecular biology tools, industrial enzymes, and models for exploring structure-function relationships. However, these uses do not often yield insight into the physiological roles of these enzymes. Although ␤-galactosidases (EC 3.2.1.23) are also found in glycoside hydrolase families (GHFs) 1, 2, and 35 (groups differing in secondary structure and other attributes [20,21]), we were especially curious about the in vivo function of GHF 42 ␤-galactosidases because they occur in organisms isolated from diverse habitats where lactose would not be present, e.g., hot springs (33, 47, 65), hypersaline environments (27, 54), and soil (Bacillus and Streptomyces spp.).Over a dozen GHF 42 enzymes have been characterized, and most are ␤-galactosidases (as shown by maximal activity with the) with less than 10% relative activity on nongalactosidic chromogens (with two exceptions [27,34]). However, experimental data supporting lactose hydrolysis by GHF 42 ␤-galactosidases are absent (34, 44, 48) or weak. Several prokaryotes possessing a GHF 42 gene are unable to grow on lactose as a sole carbon source (1, 9, 27), and at least two GHF 42 ␤-galactosidases do not cleave lactose in vitro (27,64). The determination of growth on lactose can be complicated by the presence of multiple ␤-galactosidases (7,16,25) because not all of the ␤-galactosidases may be participating equally (or at all) as...

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“…The A. niger enzyme liberated galactose and ␤-1,6-galactobiose from acid-treated larch wood and Norway spruce arabinogalactans (18). When the enzyme hydrolyzed black liquor galactan I, galactose and ␤-1,6-galactobiose were the main products, with a small amount of arabinose.…”

Section: Vol 74 2008 Endo-␤-16-galactanase From Streptomyces Avermmentioning

confidence: 99%

Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893

Ichinose

Kotake

Tsumuraya

et al. 2008

Appl Environ Microbiol

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The putative endo-␤-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of ␤-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of ␤-1,6-linked galacto-oligosaccharides, predominantly ␤-1,6-galactobiose, from ␤-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-␤-1,6-galactanase from a prokaryote.Actinomycetes are among the most numerous and ubiquitous soil bacteria. In addition to their broad range of metabolic abilities, they are important for carbon recycling in soil environments. These gram-positive bacteria are characterized by their complex morphological differentiation, resembling that of filamentous fungi, and the ability to produce a wide variety of secondary metabolites (3). Actinomycetes have been previously described as rhizosphere-colonizing bacteria (20). Plant root exudates stimulate rhizosphere growth of actinomycetes that are strongly antagonistic to fungal pathogens, while the actinomycetes utilize root exudates for growth and synthesis of antimicrobial substances (4, 27).Arabinogalactan proteins (AGPs) are found in tree exudate gums and coniferous woods (1). In addition to the complexity of protein components, the AGP molecule contains complex branched glycans (15,19,21), which are 90 to 99% of the AGP mass. Type II arabinogalactan and short oligoarabinosides are the two types of carbohydrate attached to the AGP backbone. Type II arabinogalactans have ␤-1,3-linked galactosyl backbones (sugars in the present study are D series unless otherwise designated) in mono-or oligo-␤-1,6-galactosyl and/or -arabinosyl side chains (6, 7). L-Arabinose and lesser amounts of other auxiliary sugars, such as glucuronic acid, L-rhamnose, and L-fucose, are attached to the side chains primarily at nonreducing termini (6). Glycoside hydrolases capable of degrading the carbohydrate moieties of AGPs would be a significant advance; however, there has been limited research on such enzymes.Two kinds of galactanases, exo-␤-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) and endo-␤-1,6-galactanase from Trichoderma viride (Tv1,6Gal5A, formerly Tv6GAL) were cloned for the first time (12, 16). Exo-␤-1,3-galactanases degrade ␤-1,3-galactan backbones of AGPs and belong to glycoside hydrolase (GH) family 43 (GH43) (Carbohydrate Active Enzymes database, http://www.cazy.org [8][9][10]). This enzyme was also cloned from Clostridium thermocellum and Arabidopsis thaliana, demonstrating a wide distribution including fungi, bacteria, and plants (...

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Purification and characterization of Aspergillus β-d-galactanases acting on β-1,4- and β-1,3/6-linked arabinogalactans

Cited by 32 publications

References 43 publications

Carbohydrate structures in residual lignin-carbohydrate complexes of spruce and pine pulp

Carbohydrate structures in residual lignin-carbohydrate complexes of spruce and pine pulp

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893

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